Dynamic RBM47 ISGylation confers broad immunoprotection against lung injury and tumorigenesis via TSC22D3 downregulation

ISGylation is a well-established antiviral mechanism, but its specific function in immune and tissue homeostasis regulation remains elusive. Here, we reveal that the RNA-binding protein RBM47 undergoes phosphorylation-dependent ISGylation at lysine 329 to regulate immune activation and maintain lung homeostasis. K329R knockin (KI) mice with defective RBM47-ISGylation display heightened susceptibility to LPS-induced acute lung injury and lung tumorigenesis, accompanied with multifaceted immunosuppression characterized by elevated pro-inflammatory factors, reduced IFNs/related chemokines, increased myeloid-derived suppressor cells, and impaired tertiary lymphoid structures. Mechanistically, RBM47-ISGylation regulation of the expression of TSC22D3 mRNA, a glucocorticoid-inducible transcription factor, partially accounts for the effects of RBM47-ISGylation deficiency due to its broad immunosuppressive activity. We further demonstrate the direct inhibitory effect of RBM47-ISGylation on TSC22D3 expression in human cells using a nanobody-targeted E3 ligase to induce site-specific ISGylation. Furthermore, epinephrine-induced S309 phosphorylation primes RBM47-ISGylation, with epinephrine treatment exacerbating dysregulated cytokine expression and ALI induction in K329R KI mice. Our findings provide mechanistic insights into the dynamic regulation of RBM47-ISGylation in supporting immune activation and maintaining lung homeostasis.


RNA-Seq
Lung tissue samples were obtained from 8-week-old WT and R/R littermate mice and used for RNA-sequencing.The total RNA was extracted utilizing Trizol reagent as per the manufacturer's instructions (Thermo Fisher Scientific).Each sample group comprised three biological replicates.According to the manufacturer's protocol (Illumina), stranded paired-end RNA sequencing (RNAseq) with 150 bp read lengths was performed using a HiSeq2500.The acquired sequence reads were aligned to the mouse genome sequence (mm10) by HISAT2 (version 2.1.0) 5.The number of reads per gene was determined using feature Counts v1.6.2 (WT vs. R/R samples) 6 .Differentially expressed genes were determined using edgeR 7 .GSEA analysis were performed using the clusterprofiler 8 .MCODE analysis for protein-protein interaction enrichment was performed using the Metascape online tool (www.metascape.org) 9 .

LPS-induced lung injury
Intraperitoneal injections were administered to mice utilizing lipopolysaccharide (LPS) (from E. coli O111:B6 sigma) dissolved in saline, with doses of 1 mg/kg or 2 mg/kg, and the same volume of saline was utilized as a control.At the end of 24 hours after the intraperitoneal injection, all mice were euthanized via inhalation of isoflurane, following which their lungs were extracted for further experimentation.Mice were subjected to nebulized LPS or vehicle only (control group), as previously described 10 .Briefly, the animals were exposed to nebulized LPS or saline for the vehicle control, and lung tissue was collected 24 hours (LPS 24h) after the nebulized LPS-induced lung injury.The nebulization protocol involved the administration of a total volume of 5 mL of LPS solution at a concentration of 3 mg/mL (Lipopolysaccharides from Escherichia coli 0111:B4 -Sigma-Aldrich), with sufficient time for complete solution nebulization (mean of 30 minutes).The control group received 5 mL of saline solution containing 0.9% NaCl.

Mouse model of Lewis lung carcinoma
The mouse Lewis lung cancer cell line (LLC) was intravenously administered to C57BL/6 mice at a dose of 0.25×10 6 cells per mouse via the tail vein.After three weeks of the injection, the mice were humanely euthanized by inhalation of isoflurane and the lungs were carefully extracted for further experiments.

RT-qPCR analysis
Reverse transcription was conducted using 1 µg of total RNA and ABScript II cDNA First-Strand Synthesis Kit (ABclonal) as per the manufacturer's instructions.The primer sets employed in the study have been listed in Supplementary Table S5.Real-time quantitative reverse transcriptase PCR (RT-qPCR) was utilized to measure the mRNA expression of cytokines.The RT-qPCR was conducted following the Vazyme, ChamQ Universal SYBR® qPCR Master MIX protocol (vazyme).

Production of adeno-associated viruses
The production of AAV5-shTSC22D3 and control AAV5-shNC strains was performed by Cyagen (Suzhou, China) and involved screening of DNA sequencing.The AAV plasmid was subsequently packaged into AAV serotype 5 virus by Cyagen (Suzhou, China).Following production, AAV5-shTSC22D3 (8.21 × 10 12 GC/ml) or control AAV5-shNC (7.84 × 10 12 GC/ml) were administered to the lungs via tracheal intubation and mice were treated for 6 weeks.The viral titer is expressed as Genomic Copies per mL (GC/ml).

The construction of a synthetic yeast display nanobody library
The construction of the nanobody library followed the previously described protocol. 13The DNA library pool of nanobodies was amplified for yeast transformations, and a yeast library was subsequently generated with a capacity of 2.5×10 9 .The peptide utilized for RBM47 nanobody screening was a K329 spanning peptide with the sequence CKEQYSRYQKAA.The Bovine Serum Albumin (BSA) fused peptide was diluted in FACS buffer (PBS, pH 7.4, 10 mM EDTA) and preincubated with anti-BSA antibody (mouse, 1:200) for 1 hour at room temperature.After galactose induction of nanobodies, 1×10 10 yeast cells were resuspended in FACS buffer and incubated with pretreated BSA-peptide for 1 hour at room temperature, followed by washing with FACS buffer.Yeast displaying nanobodies were incubated with anti-myc antibody, labeled with anti-rabbit IgG-Alexa Fluor 647 (1:200), and pretreated BSA-peptide was labeled with anti-mouse IgG-FITC (1:50).Yeast populations carrying dual fluorescence of FITC and Alexa Fluor 647 were enriched by flow cytometry with unlabeled yeast cells as negative control.Four rounds of flow cytometry screening were performed with sequential ten-fold decreases in antigen (BSA-peptide) concentration from 10μM to 10pM.The CDR (complementarity-determining region) sequence of nanobody nbRBM47 is ……GRTFSSYA(CDR1)……SRGGSRTN(CDR2)……RYGDYPSSSASYYNY (CDR3)…… Flow Cytometry Single-cell suspensions of lungs were prepared according to previously described methods. 14After euthanizing the mice, lung tissues were rinsed with PBS, digested with 1.5 mg/mL Collagenase A and 0.4 mg/mL DNaseI, and then filtered to create a single-cell suspension.Red blood cell fragments were treated with a lysis solution.After cell counting, 1x10 6 cells per sample were resuspended with 100ul FC flow buffer (Thermo Fisher).CD16/CD32 antibody (eBioscience) was added and incubated at 4°C for 10 min.After washing, cells were stained with a panel of standard immunophenotyping antibodies for 30 min at room temperature.Following staining, cells were washed and fixed with 0.4% paraformaldehyde in PBS.Data were acquired using a Cytoflex-LX flow cytometer and compensation was performed at the beginning of each experiment.Data were analyzed using FlowJo v10 software.

Supplementary Tables
Table S1     (lung cancer), HCT116 (colorectal cancer), CAL27 (tongue cancer), and HepG2 (liver cancer) followed by WB analysis using antibodies recognized RBM47 and Tubulin.(S1B) Immunoprecipitation (IP) coupled with mass spectrometry revealed that RBM47 interacted with ubiquitin proteasomal system (UPS) proteins.FLAG-RBM47 was expressed in 293T cells for 48h, followed by IP with anti-FLAG magnetic beads subjected to mass spectrometry (IP-MS).
Protein profile data for RBM47 interaction were shown.P indicated number of peptides identified by IP-MS, and UP indicated number of unique peptides (S1B upper panel).Protein from Ubiquitin-Proteasome System Interacting with RBM47 as Identified in the BioGRID Database (S1B lower panel).(S1C) LPS induced the ISGylation of RBM47.A549 cells were treated with 100 ng/ml LPS for 3 or 6 h followed by WB analysis using antibodies recognized RBM47 and Tubulin.(S1D) A549 cells were treated with different concentrations of LPS (0, 50, 100 ng/ml) for 6 h followed by WB analysis using antibodies recognized RBM47 and Tubulin.(S1E, S1F) Depletion of UBE1L (S1E) and UBCH8 (S1F) inhibited ISGylation of RBM47.

Primers are provided by https://www.origene.com/ Figure Legends for Supplementary Figures Supplemental Figure.1 RBM47 is ISGylated at Lys329
(S1A) Detection of RBM47 Protein Expression in four different Human Cancer Cell Lines: A549